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BioAssay: AID 588803

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assay

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assay. ..more
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AID: 588803
Data Source: The Scripps Research Institute Molecular Screening Center (PME1_INH_LCMS_ABPP-SILAC)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-11-17
Hold-until Date: 2012-05-31
Modify Date: 2012-05-31

Data Table ( Complete ):           View Data Probes    View Active Data    View All Data
BioActive Compound: Chemical Probe: 1    Active: 1
Related Experiments
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AIDNameTypeProbeComment
2130Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 depositor-specified cross reference: Summary (PME-1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Confirmation assay (PME-1 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Counterscreen (LYPLA1 inhibitors)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Counterscreen (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Counterscreen confirmation (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Counterscreen confirmation (LYPLA1 inhibitors)
2291Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors, Maybridge Library)
2363Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2aScreening depositor-specified cross reference: MOA assay (Demethylation of PP2a)
2365Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Luminescence-based counterscreen assay to identify cytotoxic compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HEC 293T)
2366Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors)
2368Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) Gel Filtration AssayScreening depositor-specified cross reference: MOA assay (PME-1 inhibitors, gel filtration assay)
2369Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) InhibitionScreening depositor-specified cross reference: ABPP screen (PME-1 inhibitors)
2371Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50: Purified enzymeConfirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors, purified enzyme)
463090Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): LC-MS/MS assay to assess binding of compounds to active siteOther depositor-specified cross reference: MOA assay (PME-1 inhibitors, LC-MS assay)
463091Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HeLa)
463124Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 2Confirmatory depositor-specified cross reference: ABPP profiling dose response (PME1 inhibitors in triplicate)
463130Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 1Confirmatory depositor-specified cross reference: ABPP profiling dose response (PME1 inhibitors in triplicate)
463131Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based inhibitionScreening depositor-specified cross reference: ABPP profiling (PME1 inhibitors in singlicate)
463132Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Inhibition of PME-1-mediated demethylation of PP2AScreening depositor-specified cross reference: MOA assay (PP2a demethylation)
463146Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Fluorescence-based biochemical gel-based ABPPOther depositor-specified cross reference: ABPP profiling (PME1 inhibitors in singlicate)
463149Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther depositor-specified cross reference: ABPP profiling (PME1 inhibition and selectivity in singlicate)
588835Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) ABHD10 selectivity assayOther depositor-specified cross reference
602468Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity among cysteine-reactive proteinsOther depositor-specified cross reference
602485Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 in vivoOther depositor-specified cross reference
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).Summary2 same project related to Summary assay
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).Summary1 same project related to Summary assay
588796Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 2Confirmatory same project related to Summary assay
588801Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10Confirmatory same project related to Summary assay
588802Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 1Confirmatory same project related to Summary assay
588804Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory same project related to Summary assay
588805Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assayOther same project related to Summary assay
588806Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10Other same project related to Summary assay
588807Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity in a complex proteome for ABHD10Other same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA132630-01
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME1_INH_LCMS_ABPP-SILAC

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assay.

Description:

Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for > 90% of all serine/ threonine phosphatase activity (1). Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. PP2A, for example, typically exists as heterotrimer with diverse subunits that may combinatorially make as many as 70 different holoenzyme assemblies (2). Mutations in several of these PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor (3). Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated (4,5). PME-1 is specifically responsible for demethylation of the carboxyl terminus (6).

Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo (7). Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas (8). In order to further elucidate the role of PP2A methylation in vivo, our lab has generated mice that lack PME-1 (PME-1 (-/-) mice) by targeted gene disruption (9). Unfortunately, PME-1 deletion resulted in perinatal lethality, underscoring the importance of PME-1 but hindering our biological studies. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors.

References:

1. Oliver, C. J., Shenolikar, S. (1998). Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 3, D961-972.
2. Janssens, V., Goris, J. (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417-439.
3. Janssens, V., Goris, J., Van Hoof, C. (2005). PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34-41.
4. Chen, J., Martin, B. L., Brautigan, D. L. (1992). Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257, 1261-1264.
5. Favre, B., Zolnierowicz, S., Turowski, P., Hemmings, B. A. (1994). The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo. J. Biol. Chem. 269, 16311-16317.
6. Lee, J., Chen, Y., Tolstykh, T., Stock, J. (1996). A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. U. S. A. 93, 6043-6047. .
7. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J. B., Broach, J. R. (2000). Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 19, 5672-5681.
8. Puustinen, P., Junttila, M. R., Vanhatupa, S., Sablina, A. A., Hector, M. E., Teittinen, K., Raheem, O., Ketola, K., Lin, S., Kast, J., Haapasalo, H., Hahn, W. C., Westermarck, J. (2009). PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 69, 2870-2877.
9. Ortega-Gutierrez, S., Leung, D., Ficarro, S., Peters, E. C., Cravatt, B. F. (2008). Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS ONE 3, e2486.

Keywords:

late stage, late stage AID, assay provider, powders, PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2A, methylation, demethylation, counterscreen, liquid chromatography, LC, tandem mass spectrometry, MS/MS, activity-based protein profiling, ABPP, stable isotope labeling with amino acids in cell culture, SILAC, ABPP-SILAC, fluorophosphonate biotin, FP-Rh, inhibitor, selectivity, anti-targets, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1Pme11protein phosphatase methylesterase 1 [Mus musculus] [gi:30794138]
Taxonomy id: 10090
Gene id: 72590
2Abhd101abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus] [gi:269784760]
Taxonomy id: 10090
Gene id: 213012
3Abhd111abhydrolase domain-containing protein 11 isoform 1 [Mus musculus] [gi:21644577]
Taxonomy id: 10090
Gene id: 68758
4Abhd121monoacylglycerol lipase ABHD12 [Mus musculus] [gi:159110817]
Taxonomy id: 10090
Gene id: 76192
5Abhd61monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]
Taxonomy id: 10090
Gene id: 66082
6Ache1acetylcholinesterase precursor [Mus musculus] [gi:13928664]
Taxonomy id: 10090
Gene id: 11423
7Acot11acyl-coenzyme A thioesterase 1 [Mus musculus] [gi:6753550]
Taxonomy id: 10090
Gene id: 26897
8Acot21acyl-coenzyme A thioesterase 2, mitochondrial precursor [Mus musculus] [gi:238624114]
Taxonomy id: 10090
Gene id: 171210
9Acot71cytosolic acyl coenzyme A thioester hydrolase isoform 1 [Mus musculus] [gi:225690616]
Taxonomy id: 10090
Gene id: 70025
10Acot91acyl-coenzyme A thioesterase 9, mitochondrial [Mus musculus] [gi:31980998]
Taxonomy id: 10090
Gene id: 56360
11Apeh1Acylpeptide hydrolase [Mus musculus] [gi:19343726]
Taxonomy id: 10090
Gene id: 235606
12Bat51abhydrolase domain-containing protein 16A [Mus musculus] [gi:30519896]
Taxonomy id: 10090
Gene id: 193742
13Ctsa1lysosomal protective protein isoform b [Mus musculus] [gi:84042523]
Taxonomy id: 10090
Gene id: 19025
14Ddhd21phospholipase DDHD2 [Mus musculus] [gi:254692989]
Taxonomy id: 10090
Gene id: 72108
15Dpp71dipeptidyl peptidase 2 precursor [Mus musculus] [gi:31981425]
Taxonomy id: 10090
Gene id: 83768
16Dpp81dipeptidyl peptidase 8 [Mus musculus] [gi:31542571]
Taxonomy id: 10090
Gene id: 74388
17Dpp91dipeptidyl peptidase 9 [Mus musculus] [gi:255003757]
Taxonomy id: 10090
Gene id: 224897
18Faah1fatty acid amide hydrolase [Mus musculus] [gi:123253900]
Taxonomy id: 10090
Gene id: 14073
19Fam108b11abhydrolase domain-containing protein FAM108B1 precursor [Mus musculus] [gi:38142456]
Taxonomy id: 10090
Gene id: 226016
20Fasn1fatty acid synthase [Mus musculus] [gi:93102409]
Taxonomy id: 10090
Gene id: 14104
21Htra21serine protease HTRA2, mitochondrial [Mus musculus] [gi:254281222]
Taxonomy id: 10090
Gene id: 64704
22Iah11isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus] [gi:27754071]
Taxonomy id: 10090
Gene id: 67732
23Lypla11acyl-protein thioesterase 1 [Mus musculus] [gi:6678760]
Taxonomy id: 10090
Gene id: 18777
24Lypla21acyl-protein thioesterase 2 [Mus musculus] [gi:7242156]
Taxonomy id: 10090
Gene id: 26394
25Lyplal11lysophospholipase-like protein 1 [Mus musculus] [gi:227496223]
Taxonomy id: 10090
Gene id: 226791
26Nceh11neutral cholesterol ester hydrolase 1 [Mus musculus] [gi:30520239]
Taxonomy id: 10090
Gene id: 320024
27Pafah1b21platelet-activating factor acetylhydrolase IB subunit beta [Mus musculus] [gi:40254624]
Taxonomy id: 10090
Gene id: 18475
28Pafah1b31platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus] [gi:6679201]
Taxonomy id: 10090
Gene id: 18476
29Pafah21platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus] [gi:225579137]
Taxonomy id: 10090
Gene id: 100163
30Parl1presenilins-associated rhomboid-like protein, mitochondrial precursor [Mus musculus] [gi:54261813]
Taxonomy id: 10090
Gene id: 381038
31Pgap11GPI inositol-deacylase [Mus musculus] [gi:254028203]
Taxonomy id: 10090
Gene id: 241062
32Pla2g151group XV phospholipase A2 precursor [Mus musculus] [gi:19527008]
Taxonomy id: 10090
Gene id: 192654
33Pla2g6185 kDa calcium-independent phospholipase A2 isoform 2 [Mus musculus] [gi:312222739]
Taxonomy id: 10090
Gene id: 53357
34Pnpla81calcium-independent phospholipase A2-gamma [Mus musculus] [gi:118130807]
Taxonomy id: 10090
Gene id: 67452
35Ppt21lysosomal thioesterase PPT2 precursor [Mus musculus] [gi:9506985]
Taxonomy id: 10090
Gene id: 54397
36Prcp1lysosomal Pro-X carboxypeptidase precursor [Mus musculus] [gi:33469015]
Taxonomy id: 10090
Gene id: 72461
37Prep1prolyl endopeptidase [Mus musculus] [gi:6755152]
Taxonomy id: 10090
Gene id: 19072
38Prepl1prolyl endopeptidase-like isoform a [Mus musculus] [gi:254939518]
Taxonomy id: 10090
Gene id: 213760
39Rbbp91putative hydrolase RBBP9 [Mus musculus] [gi:86439977]
Taxonomy id: 10090
Gene id: 26450
40Serhl1serine hydrolase-like protein [Mus musculus] [gi:13443008]
Taxonomy id: 10090
Gene id: 68607

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to determine the selectivity profile of powder samples of test compounds using activity-based protein profiling (ABPP) in combination with stable isotope labeling with amino acids in cell culture (SILAC). In this assay, cultured Neuro-2A cells are metabolically labeled with light or heavy amino acids. Light and heavy cells are treated with test compound and DMSO, respectively, in situ. Cells are lysed, proteomes are treated with the serine-hydrolase-specific activity-based fluorophosphonate-biotin (FP-biotin) affinity probe, and combined in a 1:1 (w/w) ratio. Biotinylated proteins are enriched, trypsinized, and analyzed by multi-dimensional liquid chromatography tandem mass spectrometery LC/LC-MS/MS (MudPIT). Inhibition of target and anti-target activity is quantified by comparing intensities of light and heavy peptide peaks. As designed, compounds that act as inhibitors will block FP-biotin labeling, reducing enrichment in the inhibitor-treated (light) sample relative to the DMSO-treated (heavy) sample, resulting in a smaller light/heavy ratio for each protein. Proteins not targeted by inhibitors would be expected to have a ratio of 1.

Protocol Summary:

Stable isotope labeling with amino acids in cell culture (SILAC). Neuro-2A murine neuroblastoma cells were initially grown for 10 passages in either light or heavy SILAC DMEM medium supplemented with 10% dialyzed FCS and 2 mM L-glutamine. Light media was supplemented with 100 ug/mL L-arginine and 100 ug/mL L-lysine. Heavy media was supplemented with 100 ug/mL [13C615N4]-L-Arginine and 100 ug/mL [13C615N2]-L-Lysine. Light cells (in 10 mL media) were treated with 100 nM test compound (75 uL of a 200x stock in DMSO) and heavy cells were treated with DMSO (75 uL) for 2 hours at 37 C. Cells were washed 2 times with DPBS, harvested, and homogenized by sonication in DPBS (1mL). The soluble and membrane fractions were isolated by centrifugation (100K x g, 45 minutes) and the protein concentration was adjusted to 2 mg/mL with DPBS in each fraction.

Sample preparation for ABPP-SILAC. The light and heavy proteomes were labeled with 10 uM of FP-biotin (500 uL total reaction volume) for 2 hours at 25 C. After incubation, light and heavy proteomes were mixed in 1:1 ratio, and the membrane proteomes were additionally solubilized with 1% Triton-X100. The proteomes were desalted over PD-10 desalting columns (GE Healthcare) and FP-labeled proteins were enriched with streptavidin beads. The beads were washed with 1% SDS in DPBS (1x), 6M urea (1x), and DPBS (2x), then resuspended in 6 M urea, reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30 minutes at 25 C in the dark. On-bead digestions were performed for 12 hours at 37 C with sequence-grade modified trypsin (Promega; 2 ug) in 2M urea in the presence of 2 mM CaCl2. Peptide samples were acidified to a final concentration of 5% (v/v) formic acid, pressure-loaded on to a biphasic (strong cation exchange/reversed phase) capillary column and analyzed as described below.

LC-MS/MS analysis. Digested and acidified peptide mixtures were analyzed by two-dimensional liquid chromatography (2D-LC) separation in combination with tandem mass spectrometry using an Agilent 1200-series quaternary pump and Thermo Scientific LTQ-Orbitrap Velos ion trap mass spectrometer. Peptides were eluted in a 5-step MudPIT experiment using 0%, 25%, 50%, 80%, and 100% salt bumps of 500 mM aqueous ammonium acetate and data were collected in data-dependent acquisition mode with dynamic exclusion turned on (20 s, repeat of 1). Specifically, one full MS (MS1) scan (400-1800 m/z) was followed by 30 MS2 scans of the most abundant ions. The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.9.2; publicly available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the ProLuCID algorithm (publicly available at http://fields.scripps.edu/downloads.php) against the latest version of the mouse IPI database concatenated with the reversed database for assessment of false-discovery rates. ProLucid searches allowed for static modification of cysteine residues (+57.02146 due to alkylation), methionine oxidation (+15.9949), mass shifts of labeled amino acids (+10.0083 R, 8.0142 K) and no enzyme specificity. The resulting MS2 spectra matches were assembled into protein identifications and filtered using DTASelect (version 2.0) using the --modstat, --mass, and --trypstat options (applies different statistical models for the analysis of high resolution masses, peptide digestion state, and methionine oxidation state respectively). Ratios of Light/Heavy peaks were calculated using in-house software and normalized at the peptide level to the average ratio of all non-serine hydrolase peptides. Reported ratios represent the mean of all unique, quantified peptides per protein and do not include peptides that were greater than 3 standard deviations from the median peptide value. Proteins with less than three peptides per protein ID were not included in the analysis.

Ratio = Average( AUC_light / AUC_heavy ) calculated for all unique peptides

Where:

AUC_light is the area-under-the-curve for the light peptide pair from cells treated with test compound.
AUC_heavy is the area-under-the-curve for the heavy peptide pair from cells treated with DMSO.

PubChem Activity Outcome and Score:

The following applies to each panel:

A compound with a light/heavy ratio of less than or equal to 0.5 for a particular target/anti-target was considered "active". A compound with a light/heavy ratio of greater than 0.5 for a specified target/anti-target was considered "inactive".

Overall Outcome and Score:

A compound was considered active if it was active for PME1 and inactive for all anti-target serine hydrolases tested.

The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

Neuro-2A murine cells (provided by Assay Provider)
SILAC DMEM media (Thermo 89985)
dialyzed FCS (Gemini 100-108)
1x Glutamine (CellGro 25-005-CI)
L-Arginine (SigmaAldrich A6969)
L-Lysine (SigmaAldrich L9037)
[13C615N4]-L-Arginine (SigmaAldrich 608033)
[13C615N2]-L-Lysine (SigmaAldrich 608041)
DPBS (Cellgro 20-031-CV)
FP-biotin (provided by Assay Provider)
PD-10 desalting columns (GE Healthcare 17-0851-01)
SDS (SigmaAldrich L6026)
Urea (Fisher U15-3)
TCEP (SigmaAldrich 75259)
Iodoacetamide (SigmaAldrich I1149)
Trypsin (Promega V5111)
CaCl2 (SigmaAldrich C1016)
Streptavidin beads (Pierce 20349)
Fused-silica (Agilent 160-2635-10)
Strong cation exchange (Phenomenex CH0-2257)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Millipore-filtered Water
Formic acid (Fluka 06440)
Triton-X100 (Fisher AC21568-0010)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: selectivity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Cell-based
Assay Cell Type: Neuro-2A
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [Pme1]One of Active, Inactive, or Not Tested1protein phosphatase methylesterase 1 [Mus musculus]Outcome
2Mean Ratio [Pme1]Calculated inhibitor-treated/DMSO ratio for Pme1 as mean for all unique peptides quantified1Floatratio
3Standard Error [Pme1]Calculated standard error for mean1Float
4Outcome [Abhd10]One of Active, Inactive, or Not Tested2abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus]Outcome
5Mean Ratio [Abhd10]Calculated inhibitor-treated/DMSO ratio for Abhd10 as mean for all unique peptides quantified2Floatratio
6Standard Error [Abhd10]Calculated standard error for mean2Float
7Outcome [Abhd11]One of Active, Inactive, or Not Tested3abhydrolase domain-containing protein 11 isoform 1 [Mus musculus]Outcome
8Mean Ratio [Abhd11]Calculated inhibitor-treated/DMSO ratio for Abhd11 as mean for all unique peptides quantified3Floatratio
9Standard Error [Abhd11]Calculated standard error for mean3Float
10Outcome [Abhd12]One of Active, Inactive, or Not Tested4monoacylglycerol lipase ABHD12 [Mus musculus]Outcome
11Mean Ratio [Abhd12]Calculated inhibitor-treated/DMSO ratio for Abhd12 as mean for all unique peptides quantified4Floatratio
12Standard Error [Abhd12]Calculated standard error for mean4Float
13Outcome [Abhd6]One of Active, Inactive, or Not Tested5monoacylglycerol lipase ABHD6 [Mus musculus]Outcome
14Mean Ratio [Abhd6]Calculated inhibitor-treated/DMSO ratio for Abhd6 as mean for all unique peptides quantified5Floatratio
15Standard Error [Abhd6]Calculated standard error for mean5Float
16Outcome [Ache]One of Active, Inactive, or Not Tested6acetylcholinesterase precursor [Mus musculus]Outcome
17Mean Ratio [Ache]Calculated inhibitor-treated/DMSO ratio for Ache as mean for all unique peptides quantified6Floatratio
18Standard Error [Ache]Calculated standard error for mean6Float
19Outcome [Acot1]One of Active, Inactive, or Not Tested7acyl-coenzyme A thioesterase 1 [Mus musculus]Outcome
20Mean Ratio [Acot1]Calculated inhibitor-treated/DMSO ratio for Acot1 as mean for all unique peptides quantified7Floatratio
21Standard Error [Acot1]Calculated standard error for mean7Float
22Outcome [Acot2]One of Active, Inactive, or Not Tested8acyl-coenzyme A thioesterase 2, mitochondrial precursor [Mus musculus]Outcome
23Mean Ratio [Acot2]Calculated inhibitor-treated/DMSO ratio for Acot2 as mean for all unique peptides quantified8Floatratio
24Standard Error [Acot2]Calculated standard error for mean8Float
25Outcome [Acot7]One of Active, Inactive, or Not Tested9cytosolic acyl coenzyme A thioester hydrolase isoform 1 [Mus musculus]Outcome
26Mean Ratio [Acot7]Calculated inhibitor-treated/DMSO ratio for Acot7 as mean for all unique peptides quantified9Floatratio
27Standard Error [Acot7]Calculated standard error for mean9Float
28Outcome [Acot9]One of Active, Inactive, or Not Tested10acyl-coenzyme A thioesterase 9, mitochondrial [Mus musculus]Outcome
29Mean Ratio [Acot9]Calculated inhibitor-treated/DMSO ratio for Acot9 as mean for all unique peptides quantified10Floatratio
30Standard Error [Acot9]Calculated standard error for mean10Float
31Outcome [Apeh]One of Active, Inactive, or Not Tested11Acylpeptide hydrolase [Mus musculus]Outcome
32Mean Ratio [Apeh]Calculated inhibitor-treated/DMSO ratio for Apeh as mean for all unique peptides quantified11Floatratio
33Standard Error [Apeh]Calculated standard error for mean11Float
34Outcome [Bat5]One of Active, Inactive, or Not Tested12abhydrolase domain-containing protein 16A [Mus musculus]Outcome
35Mean Ratio [Bat5]Calculated inhibitor-treated/DMSO ratio for Bat5 as mean for all unique peptides quantified12Floatratio
36Standard Error [Bat5]Calculated standard error for mean12Float
37Outcome [Ctsa]One of Active, Inactive, or Not Tested13lysosomal protective protein isoform b [Mus musculus]Outcome
38Mean Ratio [Ctsa]Calculated inhibitor-treated/DMSO ratio for Ctsa as mean for all unique peptides quantified13Floatratio
39Standard Error [Ctsa]Calculated standard error for mean13Float
40Outcome [Ddhd2]One of Active, Inactive, or Not Tested14phospholipase DDHD2 [Mus musculus]Outcome
41Mean Ratio [Ddhd2]Calculated inhibitor-treated/DMSO ratio for Ddhd2 as mean for all unique peptides quantified14Floatratio
42Standard Error [Ddhd2]Calculated standard error for mean14Float
43Outcome [Dpp7]One of Active, Inactive, or Not Tested15dipeptidyl peptidase 2 precursor [Mus musculus]Outcome
44Mean Ratio [Dpp7]Calculated inhibitor-treated/DMSO ratio for Dpp7 as mean for all unique peptides quantified15Floatratio
45Standard Error [Dpp7]Calculated standard error for mean15Float
46Outcome [Dpp8]One of Active, Inactive, or Not Tested16dipeptidyl peptidase 8 [Mus musculus]Outcome
47Mean Ratio [Dpp8]Calculated inhibitor-treated/DMSO ratio for Dpp8 as mean for all unique peptides quantified16Floatratio
48Standard Error [Dpp8]Calculated standard error for mean16Float
49Outcome [Dpp9]One of Active, Inactive, or Not Tested17dipeptidyl peptidase 9 [Mus musculus]Outcome
50Mean Ratio [Dpp9]Calculated inhibitor-treated/DMSO ratio for Dpp9 as mean for all unique peptides quantified17Floatratio
51Standard Error [Dpp9]Calculated standard error for mean17Float
52Outcome [Faah]One of Active, Inactive, or Not Tested18fatty acid amide hydrolase [Mus musculus]Outcome
53Mean Ratio [Faah]Calculated inhibitor-treated/DMSO ratio for Faah as mean for all unique peptides quantified18Floatratio
54Standard Error [Faah]Calculated standard error for mean18Float
55Outcome [Fam108b1]One of Active, Inactive, or Not Tested19abhydrolase domain-containing protein FAM108B1 precursor [Mus musculus]Outcome
56Mean Ratio [Fam108b1]Calculated inhibitor-treated/DMSO ratio for Fam108b1 as mean for all unique peptides quantified19Floatratio
57Standard Error [Fam108b1]Calculated standard error for mean19Float
58Outcome [Fasn]One of Active, Inactive, or Not Tested20fatty acid synthase [Mus musculus]Outcome
59Mean Ratio [Fasn]Calculated inhibitor-treated/DMSO ratio for Fasn as mean for all unique peptides quantified20Floatratio
60Standard Error [Fasn]Calculated standard error for mean20Float
61Outcome [Htra2]One of Active, Inactive, or Not Tested21serine protease HTRA2, mitochondrial [Mus musculus]Outcome
62Mean Ratio [Htra2]Calculated inhibitor-treated/DMSO ratio for Htra2 as mean for all unique peptides quantified21Floatratio
63Standard Error [Htra2]Calculated standard error for mean21Float
64Outcome [Iah1]One of Active, Inactive, or Not Tested22isoamyl acetate-hydrolyzing esterase 1 homolog [Mus musculus]Outcome
65Mean Ratio [Iah1]Calculated inhibitor-treated/DMSO ratio for Iah1 as mean for all unique peptides quantified22Floatratio
66Standard Error [Iah1]Calculated standard error for mean22Float
67Outcome [Lypla1]One of Active, Inactive, or Not Tested23acyl-protein thioesterase 1 [Mus musculus]Outcome
68Mean Ratio [Lypla1]Calculated inhibitor-treated/DMSO ratio for Lypla1 as mean for all unique peptides quantified23Floatratio
69Standard Error [Lypla1]Calculated standard error for mean23Float
70Outcome [Lypla2]One of Active, Inactive, or Not Tested24acyl-protein thioesterase 2 [Mus musculus]Outcome
71Mean Ratio [Lypla2]Calculated inhibitor-treated/DMSO ratio for Lypla2 as mean for all unique peptides quantified24Floatratio
72Standard Error [Lypla2]Calculated standard error for mean24Float
73Outcome [Lyplal1]One of Active, Inactive, or Not Tested25lysophospholipase-like protein 1 [Mus musculus]Outcome
74Mean Ratio [Lyplal1]Calculated inhibitor-treated/DMSO ratio for Lyplal1 as mean for all unique peptides quantified25Floatratio
75Standard Error [Lyplal1]Calculated standard error for mean25Float
76Outcome [Nceh1]One of Active, Inactive, or Not Tested26neutral cholesterol ester hydrolase 1 [Mus musculus]Outcome
77Mean Ratio [Nceh1]Calculated inhibitor-treated/DMSO ratio for Nceh1 as mean for all unique peptides quantified26Floatratio
78Standard Error [Nceh1]Calculated standard error for mean26Float
79Outcome [Pafah1b2]One of Active, Inactive, or Not Tested27platelet-activating factor acetylhydrolase IB subunit beta [Mus musculus]Outcome
80Mean Ratio [Pafah1b2]Calculated inhibitor-treated/DMSO ratio for Pafah1b2 as mean for all unique peptides quantified27Floatratio
81Standard Error [Pafah1b2]Calculated standard error for mean27Float
82Outcome [Pafah1b3]One of Active, Inactive, or Not Tested28platelet-activating factor acetylhydrolase IB subunit gamma [Mus musculus]Outcome
83Mean Ratio [Pafah1b3]Calculated inhibitor-treated/DMSO ratio for Pafah1b3 as mean for all unique peptides quantified28Floatratio
84Standard Error [Pafah1b3]Calculated standard error for mean28Float
85Outcome [Pafah2]One of Active, Inactive, or Not Tested29platelet-activating factor acetylhydrolase 2, cytoplasmic [Mus musculus]Outcome
86Mean Ratio [Pafah2]Calculated inhibitor-treated/DMSO ratio for Pafah2 as mean for all unique peptides quantified29Floatratio
87Standard Error [Pafah2]Calculated standard error for mean29Float
88Outcome [Parl]One of Active, Inactive, or Not Tested30presenilins-associated rhomboid-like protein, mitochondrial precursor [Mus musculus]Outcome
89Mean Ratio [Parl]Calculated inhibitor-treated/DMSO ratio for Parl as mean for all unique peptides quantified30Floatratio
90Standard Error [Parl]Calculated standard error for mean30Float
91Outcome [Pgap1]One of Active, Inactive, or Not Tested31GPI inositol-deacylase [Mus musculus]Outcome
92Mean Ratio [Pgap1]Calculated inhibitor-treated/DMSO ratio for Pgap1 as mean for all unique peptides quantified31Floatratio
93Standard Error [Pgap1]Calculated standard error for mean31Float
94Outcome [Pla2g15]One of Active, Inactive, or Not Tested32group XV phospholipase A2 precursor [Mus musculus]Outcome
95Mean Ratio [Pla2g15]Calculated inhibitor-treated/DMSO ratio for Pla2g15 as mean for all unique peptides quantified32Floatratio
96Standard Error [Pla2g15]Calculated standard error for mean32Float
97Outcome [Pla2g6]One of Active, Inactive, or Not Tested3385 kDa calcium-independent phospholipase A2 isoform 2 [Mus musculus]Outcome
98Mean Ratio [Pla2g6]Calculated inhibitor-treated/DMSO ratio for Pla2g6 as mean for all unique peptides quantified33Floatratio
99Standard Error [Pla2g6]Calculated standard error for mean33Float
100Outcome [Pnpla8]One of Active, Inactive, or Not Tested34calcium-independent phospholipase A2-gamma [Mus musculus]Outcome
101Mean Ratio [Pnpla8]Calculated inhibitor-treated/DMSO ratio for Pnpla8 as mean for all unique peptides quantified34Floatratio
102Standard Error [Pnpla8]Calculated standard error for mean34Float
103Outcome [Ppt2]One of Active, Inactive, or Not Tested35lysosomal thioesterase PPT2 precursor [Mus musculus]Outcome
104Mean Ratio [Ppt2]Calculated inhibitor-treated/DMSO ratio for Ppt2 as mean for all unique peptides quantified35Floatratio
105Standard Error [Ppt2]Calculated standard error for mean35Float
106Outcome [Prcp]One of Active, Inactive, or Not Tested36lysosomal Pro-X carboxypeptidase precursor [Mus musculus]Outcome
107Mean Ratio [Prcp]Calculated inhibitor-treated/DMSO ratio for Prcp as mean for all unique peptides quantified36Floatratio
108Standard Error [Prcp]Calculated standard error for mean36Float
109Outcome [Prep]One of Active, Inactive, or Not Tested37prolyl endopeptidase [Mus musculus]Outcome
110Mean Ratio [Prep]Calculated inhibitor-treated/DMSO ratio for Prep as mean for all unique peptides quantified37Floatratio
111Standard Error [Prep]Calculated standard error for mean37Float
112Outcome [Prepl]One of Active, Inactive, or Not Tested38prolyl endopeptidase-like isoform a [Mus musculus]Outcome
113Mean Ratio [Prepl]Calculated inhibitor-treated/DMSO ratio for Prepl as mean for all unique peptides quantified38Floatratio
114Standard Error [Prepl]Calculated standard error for mean38Float
115Outcome [Rbbp9]One of Active, Inactive, or Not Tested39putative hydrolase RBBP9 [Mus musculus]Outcome
116Mean Ratio [Rbbp9]Calculated inhibitor-treated/DMSO ratio for Rbbp9 as mean for all unique peptides quantified39Floatratio
117Standard Error [Rbbp9]Calculated standard error for mean39Float
118Outcome [Serhl]One of Active, Inactive, or Not Tested40serine hydrolase-like protein [Mus musculus]Outcome
119Mean Ratio [Serhl]Calculated inhibitor-treated/DMSO ratio for Serh1 as mean for all unique peptides quantified40Floatratio
120Standard Error [Serhl]Calculated standard error for mean40Float
Additional Information
Grant Number: 1 R01 CA132630-01

Classification
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